Supplemental Material

This article contains the following supporting material:

• Movie 1 - Time-lapse imaging of YFP-occludin during tight junction assembly after calcium switch, in control cells.

• Movie 2 - Time-lapse imaging of YFP-occludin during tight junction assembly after calcium switch, in cells lacking ZO-1.

• Figure 1 - ZO-1 knockdown does not disrupt tight junctions. MDCK cells were transfected with either pS-Luciferase or pS-ZO-1, and pK-YFP as a transfection marker. At day 3 post-transfection, cells were fixed using MeOH/Acetone and stained with mouse anti-Occludin (red), chicken anti-Na⁺K⁺ATPase (green), and rabbit anti-GFP (blue). Secondary antibodies were Alexa546 goat anti-mouse , Alexa488 goat anti-chicken , and Alexa647 goat anti-rabbit (Molecular Probes). Images were acquired on a Ziess LSM510/META confocal microscope and processed using Improvision Volocity software. Z-stack assemblies consist of 15 x 1μm sections. XY field show the merged stacks.

• Figure 2 - Time-lapse imaging of YFP-ZO-1 during tight junction assembly after calcium switch. A stable cell line of MDCK cells that express YFP-ZO-1 was incubated overnight in medium lacking calcium. The cells were mounted on a Perkin-Elmer spinning disk confocal microscope (Nikon Eclipse T200) with a 100x lens (na 1.4). Cells were maintained at 37°C. The medium was replaced by pre-warmed F12 medium containing calcium, 20 mM Hepes buffer (pH 7.2) and Oxyrase (to reduce phototoxicity), and images were collected at a rate of 0.5 frame/min for about 2 hrs. Stacks were manipulated using Volocity software, and projected images were processed in Photoshop to increase contrast.